We are investigating the roles of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, as specific regulators of signaling in the immune system. Specifically, the family of NADPH oxidase (Nox) enzymes deliberately generates ROS in certain settings. Phagocytes produce large amounts of ROS in response to infectious or inflammatory stimuli through the prototypic NADPH oxidase containing gp91phox (Nox2). Recent discovery of multiple homologues of gp91phox (e.g., Nox1, Nox3-5, Duox1/2) has opened studies on the roles of NADPH oxidase activity in non-phagocytic cells. In non-phagocytic cells and tissues, Nox family members produce lower levels of ROS that can act as signaling molecules. We found that T cells express at least two NADPH oxidase family members and that the absence of NADPH oxidases leads to a deficiency in T cell receptor (TCR) stimulated ROS generation and altered T cell responses. [unreadable] [unreadable] T cells express a phagocyte-type NADPH oxidase dependent on Nox2 and p47phox, which is responsible for a second phase of hydrogen peroxide generation among three ROS generation phases induced by TCR stimulation. Nox2-dependent ROS increased TH2 cytokine production and inhibited TH1 cytokine generation and the mitogen-activated protein kinase (MAPK) pathway. Our new findings indicate that T cells also express the calcium-dependent NADPH oxidase Dual oxidase 1 (Duox1). Duox1 is responsible for the early phase hydrogen peroxide generation induced by TCR stimulation, which depends on protein tyrosine kinase activities, phospholipase C-gamma1 (PLC-gamma1) activity, inositol-1,4,5-triphosphate (IP3) generation through IP3 receptor and release of calcium from endoplasmic reticulum (ER) stores. Knockdown of Duox1 expression inhibited the mitogen-activated protein kinase (MAPK) pathway, Calcium influx from extracellular medium after TCR stimulation, and secretion of multiple cytokines including IL-2, IL-4, IFN-gamma, and TNF-alpha. Thus, T cells express multiple NADPH oxidases and both Nox2 and Duox1 regulate TCR signal transduction. [unreadable] [unreadable] We made remarkable progress in our understanding of the molecular nature of TCR-dependent ROS generation in T cells. With the identification of NADPH oxidase Duox1 as a regulator of TCR signal transduction, we now have the tools to study the molecular mechanism of TCR-dependent ROS generation in greater detail, including its association with calcium and other signaling pathways. We can study the role of Duox1-dependent ROS in T-cell-and B-cell-mediated immune responses in vivo, using Duox1-knockout mice, and the involvement of ROS signals in development and selection processes in thymocytes.